高溫下水稻穎果發育中miRNAs之表現及稻米品質形成相關性之研究

Studies on the Relationship between MicroRNAs and High Temperature Effects on Quality Formation in Developing Caryopsis of Rice (Oryza sativa L.)

李佳諭(Chia-Yu Li)

指導教授 ： 盧虎生

國立臺灣大學/生物資源暨農學院/農藝學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.10152

摘要

台灣一期稻作易因充實期高溫導致稻米白堊質發生，進而降低碾米產量與稻米品質。而miRNA是基因的上游調控因子，在植物生長分化過程扮演重要角色，有關其在高溫水稻穎果發育的研究甚為缺乏。為探討miRNA可能參與高溫稻米白堊質形成之生理機制，本研究內容使用兩個水稻品種進行生理與基因表現分析。
TK9是良質米品種，高溫易導致此品種白堊質增加，且已知高溫加氮可降低高溫下TK9穀粒的白堊質發生率。以TK9發育穎果進行microarray，分析常溫、高溫與高溫加氮的miRNA表現趨勢，並進行real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)分析確認miRNA的表現。結果發現36 個miRNA參與TK9發育穎果的高溫反應。再比較高溫下反應之miRNA及其可能下游調控基因，與胚乳白堊質性狀相關的QTL基因座(quantitative trait loci)區間，選擇81個下游基因進行qRT-PCR分析。結果顯示其中有30個下游基因與miRNA具有相反的表現趨勢，可能與高溫下的穎果發育有關。高溫下miR812與miR818在6-9天發育穎果促進表現，而這兩個miRNA的下游基因，citrate carrier與pex14則在高溫穎果顯示與miRNA相反的表現趨勢。由這兩個下游基因的生理功能推測早期發育穎果可能受高溫促進TCA cycle與抑制過氧化體功能。
此外，已知SA1739在高溫下具有輕微的白堊質外觀。取TNG67 (Oryza sativa L. Japonica cv. Tainung 67)及其EMS (ethyl methane sulfonate)耐熱突變株SA1739的發育穎果進行DNA microarray基因表現的轉錄體研究。總計得到6009個在高溫下具差異性表現的基因，其中7個基因被預測是5個miRNAs的下游基因。而這5個miRNAs顯示在TK9發育穎果受高溫誘導大量表現，推測這5個miRNAs可能參與高溫下的白堊質穀粒的發育。LON protease是siR806的預測下游基因，高溫抑制LON protease在SA1739發育穎果的表現，推測抑制高溫發育穎果粒腺體內的蛋白質分解活性將有利於發展穎果的耐熱性。LPP3 (lipid phosphate phosphatase 3)催化PA (phosphatidic acid)的去磷酸化反應。高溫抑制TNG67發育穎果的LPP3表現可能導致PA累積在發育穎果內，進而提高高溫下發育穎果的乙烯敏感度。
除此之外，進行與能量代謝有關的兩個基因 (pyruvate kinase and alcohol dehydrogenase) 在TK9發育穎果的表現分析，顯示高溫下兩基因出現共同促進表現的趨勢，推測高溫導致發育穎果進入缺氧的能量代謝生理。為進一步測試乙烯是否參予高溫下胚乳酸化與白堊質形成過程，取高溫生長穎果處理乙烯抑制劑(AgNO3)，結果顯示降低高溫下胚乳酸化過程與白堊質發生。綜合上述研究，本研究篩選出高溫下發育穎果的miRNA及其可能的下游調控基因，可提供進一步探討有關高溫下穎果發育過程的生理轉變和基因調控機制。這些資訊將可做為未來高溫環境下育成耐熱新品種的理論基礎。

關鍵字

微小RNA ； 高溫 ； 白堊質 ； 稻米品質 ； 氮素

並列摘要

The chalkiness of rice grain is a primary defect under high temperature stress. The building of chalky endosperm was a result of low energy status, high oxidative stress, high endosperm acidification, and high ethylene evolution within caryopsis developing under high temperature. To gain insight into the mechanisms caused the physiological and biochemical changes, the expression profiles of miRNAs under high temperature in the developing caryopsis of TK9 were studied by microarray analyses. TK9 (Oryza sativa L. Japonica cv. Taiken 9) is a heat sensitive rice variety having a higher ratio of chalky grain under high temperature. After verified by qRT-PCR (real-time quantitative reverse transcriptase-polymerase chain reaction), thirty six miRNAs were confirmed to respond to high temperature. Compared the chromosomal locations of potential miRNA targets with that of QTLs (quantitative trait loci) related to the buildup of chalky endosperm, thirty out of 81 potential target genes were confirmed to be separately down-regulated by 17 miRNAs. Under high temperature, at 6- to 9-day caryopsis, two miRNAs, miR812 and miR818, were induced, while their predicted target genes, citrate carrier and pex14, showed expected inverse patterns. The decreased expressions of citrate export (citrate carrier) from mitochondria and pex14 may result in improved TCA cycle and reduced function of peroxisome, respectively.
Moreover, the transcriptomes of a high temperature resistant mutant, SA1737, and its corresponding wild type, TNG67 (Oryza sativa L. Japonica cv. Tainung 67) was profiled by DNA microarray analyses. In total, 6,009 genes having significantly different expression levels were detected in compare with high temperature. 7 of them are the putative target genes of 5 miRNAs. These miRNAs in TK9 involving in responsiveness to high temperature indicated that miRNAs may have a regulatory role in the development of chalky grain upon high temperature. LON protease was a predicted target of siR806, suppression of LON protease in SA1737 suggested inhibition of proteolysis within mitochondria was crucial for the resistance to high temperature in developing caryopsis. LPP3 (lipid phosphate phosphatase 3), the enzyme catalyzed the dephosphorylation of PA (phosphatidic acid) was predicted to be a regulatory target of miR319. Suppression of LPP3 and accumulation of PA in TNG67 could activate ethylene signaling pathway under high temperature.
In addition, two genes (pyruvate kinase and alcohol dehydrogenase) involved in energy metabolism were examined in TK9. Both genes were co-regulated under high temperature, indicating that the energy production in developing caryopsis has been switched to the anaerobic pathway. Furthermore, to test whether ethylene accumulation in the developing caryopsis is related to the endosperm acidification and chalky occurrence under high temperature, an inhibitor of ethylene production (AgNO3) was applied to the developing caryopses of TK9 plants. As expected, the level of endosperm acidification and the ratio of chalky occurrence were reduced under high temperature. In this study, we identified miRNA-mediated regulations that responded to high temperature in developing rice grain. The data shed light on transcriptional gene regulations in chalky grain development under high temperature. The information obtained here could further be used by breeders to develop potential strategies for breeding heat-tolerant rice cultivars.

並列關鍵字

miRNA ； high temperature ； chalky grain ； grain quality ； nitrogen

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